Dat1

ABSTRACT

A synthetic diaminoketothiazole, its process of preparation and its use as a microtubule inhibitor, a probe for tubulin-microtubule system and a cytotoxic agent. Diaminoketothiazole of the formula (I) wherein Ar is 4-OMe-C 6 H 5 , Ar′ is C 6 H 5 .

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 11/578,566, filed Jan. 8, 2008 and entitled “DAT1”,which is the United States National phase application of PCT/IN04/00108,filed Apr. 16, 2004. The entire contents of the above-referencedapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to Diaminoketothiazole (DAT1) end to the processof preparation thereof.

Further this invention also relates to the use of Diaminoketothiazole(DAT1) as a microtubule inhibitor, a probe for tubulin-microtubulesystem and a cytotoxic agent.

2. Description of Related Art

Microtubules are a topic of intense research because of their importantand multiple functions in the cell. Many of the potential anticanceragents act on microtubules and arrest mitosis as during mitotic celldivision, microtubules play a crucial role by maintaining proper spindlefunction. Microtubule effectors work in two ways, they can interferewith microtubule dynamics and they can shift the tubulin-microtubuleequilibrium in the cell by either inducing or inhibiting microtubulepolymerization. There are three major classes of microtubule effectors.Taxanes stabilize microtubules by blocking disassembly. Vinca alkaloidsand colchicine site binders destabilize microtubules by the inhibitionof assembly of tubulin molecules, the major component of microtubules.Taxanes like Paclitaxel, docetaxel and vinca alkaloids like vincristineand vinblastine are well characterized and widely used clinically indifferent types of malignancies.

The main drawback of Taxanes and vinca alkaloids is that their use islimited by the development of drug resistance, neurotoxicity and limitedavailability leading to very high expenses involved.

The derivatives of diaminoketothiazoles have received much attentionlately as inhibitors of cyclin-dependent kinases and glycogen synthasekinase-3. These are thus claimed to be useful for the treatment ofmalignancies and Alzheimer's disease, impaired glucose tolerance, Type 1and 2 diabetes.

For the synthesis of diaminoketothiazoles, there exist only few methods.The first method makes use of a cyanothiourea derivative to provide the(C—N—C—S) atoms required for the thiazole construction and the remainingC atom is sourced from an alpha-haloketone. The second method utilizesthiocarbamoylamidine derivatives as the source of the (C—N—C—S)four-atom complement.

In the third approach, an S-alkyldithiobiuret serves as synthon forproviding the (C—N—C—S) four-atom complement. These methods are usuallysuited for the solution phase synthesis of the title compounds. However,in the light of combinatorial library synthesis, solid phase methods aremuch more desirable. Such approaches allow rapid synthesis of a largenumber of analogue molecules that can be later subjected to bioactivityscreening.

OBJECTS OF THE INVENTION

An object of this invention is to propose a Diaminoketothiazole (DAT1)and a novel process for the preparation thereof.

Another object of this invention is to propose a new solid phasesynthesis of diaminoketothiazoles.

Further object of this invention is to propose a useful method for thesynthesis of diaminoketothiazoles on a solid support.

Still further object of this invention is to propose a process ofsynthesis of diaminoketothiazoles (DAT1) which is cheap and costeffective.

Another object of this invention is to propose DAT1 as a microtubuleinhibitor and cytotoxic agent.

Still another object of this invention is to proposediaminoketothiazoles for the treatment of cancer and other disease usingits microtubule inhibition activity.

Yet another object of this invention is to propose diaminoketothiazolesas a probe for structure-function studies of tubulin-microtubule system.

BRIEF DESCRIPTION OF THE INVENTION

According to this invention there is provided a Diaminoketothiazole(DAT1). Further, according to this invention there is also provided aprocess for the solid phase synthesis of diaminoketothiazolescomprising, reacting aminomethylpolystyrene beads with1-[N-(arylthiocarbamoyl)amidino]-3,5-dimethylpyrazole to produceN—(N-arylthiocarbamoyl)-N-guanidinomethyl polystyrene (2); reacting saidN—(N-arylthiocarbamoyl)-N-guanidinomethyl polystyrene with alphahaloketones in the presence of a base which produces the acyclic S-alkylintermediate derivative (3); subjecting the said intermediate thusformed directly, without isolation, to the step of cyclisation to obtainthe intermediate cyclic thiazoline (4); subjecting the intermediate thusformed to a step of eliminative aromatization again directly, withoutisolation, to produce diaminoketothiazole in the solution; filtering thesaid solution to remove the polymer beads, if any, and impurities andsubsequently isolating 5-aroyl-4-amino-2-arylaminothiazoles from thesolution by precipitation.

According to another embodiment of this invention, there is provided theuse of diaminoketothiazole as a microtubule inhibitor, a tubulin bindingagent and a cytotoxic agent.

DESCRIPTION OF THE ACCOMPANYING FIGURES

FIG. 1: Time-course of inhibition of in vitro microtubule assembly byDAT1 1.2 mg/ml of 3×MTP was incubated with different concentrations ofDAT1 for 2 min at 24° C., in PEM buffer. Subsequently 1 mM GTP was addedand polymerization was followed by the turbidity at 345 nm for 20 min at37° C. Control MTP (1), MTP with 10 μM DAT1 (2), 20 μM DAT1 (3) and 40μM DAT1 (4).

FIG. 2: Effect of DAT1 on the microtubule network HeLa cells wereexposed to DMSO (A & B), 1 μM (C) or 0.2 μM (D) DAT1 and 0.1 μM (E) or0.02 μM (F) vinblastine. After 24 h, microtubules were visualized byindirect immunofluorescence microscopy using an antibody β-tubulin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the solid phase synthesis ofdiaminoketothiazoles on polymer beads. The details of the new inventionare described below.

Polymer beads comprising DVB-cross linked chloromethylpolystyrene suchas 2% by wt, was converted to aminomethylpolystyrene (AMPS) by areported method. The aminomethylpolystyrene beads so obtained were thenreacted by a new method with1-[(N-arylthiocarbamoyl)amidino]-3,5-dimethylpyrazole 1 which acts as athiocarbamoyl group transfer agent. This converts the amino group on thepolymer bead into a N—(N-arylthiocarbamoyl)guanidine group giving novelN—(N-arylthiocarbamoyl)-N′-guanidinomethyl polystyrene 2 (AGMPS) is onreaction with alpha haloketones in the presence of a base gives theacyclic S-alkyl intermediate derivative 3, which then directly and incitu undergoes a cyclisation to the next intermediate cyclic thiazoline4, followed by an eliminative aromatisation step in which theaminomethyl polystyrene acts as a leaving group, thus leading to therelease of diaminoketothiazole 5 in solution. A filtration removes thepolymer, dilution of the solvent with water cleanly precipitates theproduct 5-aroyl-4-amino-2-arylaminothiazoles 5.

Reaction Scheme

DAT1 distorts microtubules in HeLa cells as well as inhibits the invitro assembly of microtubular proteins. It exhibits cytotoxicity indifferent types of cancer cell lines and is much more active thanpaclitaxel and somewhat more active than vinblastine in drug resistantcancer cells. It is much less toxic to normal cells than cancer cells.Moreover, its synthesis is quite cheap compared to the cost involved inthe synthesis of taxanes and vinca alkaloids.

The compound DAT1 was tested for its cytotoxicity on human cervical,uterus and colon cancer cell lines and mouse fibrocercoma cells. Celllines were obtained from ATCC, USA and NCCS, Pune, India. MTT assay,which correlates a formazan dye formation with the number of viablecells, was used for this purpose. The widely used anticancer drugspaclitaxel, vinblastine or the antimitotic drug colchicine were used forcomparison. In 5 out of the 9 cell lines tested, DAT1 showed activitywith IC₅ values in a range of 0.05-0.3 μM, and in 2 cell lines, thevalues were in the range of 1-5 μM (Table 1). These values were eithercomparable or 5-20 times lower than paclitaxel and vinblastine.Subsequently, DAT1 was tested on the multidrug resistant cell lineMES-SA/DX5, which is resistant to a number of important antimitotic andanticancer agents, viz, colchicine, paclitaxel, vinca alkaloids,doxorubicin etc. It was found to be 15 and 2 times more active thanpaclitaxel and vinblastine respectively.

The cell survival in the normal immortalized cell line IMR 90 (lungepithelial) was good after the treatment of DAT1 in a concentrationwhich was much more than the IC₅₀ values in all the cancer cell linestested. In comparison, the cell survival was less upon vinblastinetreatment and similar upon paclitaxel treatment in similarconcentrations (data not shown).

As many of the potential anticancer drugs are antimitotic andmicrotubule effectors, DAT1 was tested for its effect on microtubuleassembly. A spectrophotometric assay was used for this purpose whereturbidity at 350 nm was used to quantitate amount of microtubulepolymers formed from microtubular proteins. FIG. 1 shows that itinhibited microtubule formation in a concentration dependent manner.

The in vivo effect of DAT1 on microtubules was tested on HeLa (cervicalcancer cell line) cells after an incubation of 48 hours and staining themicrotubule network by an antitubulin antibody followed by a Rhodaminelabeled secondary antibody. FIG. 2 shows that microtubule network wasdestroyed by DAT1 in a similar manner to the anticancer drug,vinblastine.

As tubulin is the major component of microtubulin the effect of DAT1 onpurified tubulin was checked. DAT1 absorbs light with absorption maximaat 212 nm, 283 nm and 374 nm in methanol. Although DAT1 doesn't exhibitany fluorescence by itself in aqueous solution, when incubated withtubulin, it showed fluorescence with an emission maximum of 457 nm uponexcitation at 374 nm. The fluorescence intensity increased with theincrease in concentration of tubulin showing that it bound to tubulin.

To measure the binding affinity and stoichiometry of DAT1 binding totubulin, a titration of tubulin with DAT1 at 24° C., was performed andthe fluorescence values at 450 nm were noted upon excitation at 374 nm.A K_(d) (Dissociation constant) value of 2.9±1 μM and a stoichiometry of1 were calculated (mean of three experiments) from a scatchard plot.

All these observations place DAT1 in a suitable position forconsideration as a good microtubule inhibitor, a suitable probe for thestructure-function studies of tubulin-microtubule system and a potentialanticancer agent. The physical (IR spectral, NMR and MS spectral) dataare shown in Table 2.

EXAMPLES 1. Conversion of aminomethylpolystyrene (AMPS) toN—(N-arylthiocarbamoyl)-N′-guanidinomethyl polystyrene 2 (AGMPS) GeneralProcedure

Aminomethylpolystyrene resin beads (2 g, 2.13 meq. NH₂/g resin) wasswelled in acetonitrile (5 ml). To the swelled resin, a solution of1-[(N-arylthiocarbamoyl)amidino]-3,5-dimethylpyrazole 1 (2 molarequivalents) in acetonitrile (10 mL) was added. The mixture was thenrefluxed for 12-15 h. The resin beads were then removed by filtration,washed repeatedly with warm and then cold acetonitrile (3×10 ml), thenwith petroleum ether (60-80° b.p) (2×10 ml) and then dried in vacuum.The S capacity of the resin was then estimated by digestion andgravimetry by standard procedures. This was found to be in the range0.98-1.32 meg/g resin.

2. Synthesis of 5-acyl-4-amino-2-arylaminothiazoles 5 General Procedure

The above arylthiocarbamoyl resin (AGMPS) was swelled in N,N-dimethylformamide (DMF) (5 ml). To this, the respective alpha-bromoketone (molarequivalent as per S-capacity) in DMF (2 ml) was added followed by twomolar equivalents of triethylamine.

The mixture was warmed to 50-60° C. for 2-5 h. The resin beads wereremoved by filtration, washed with DMF and the pooled filtrate andwashings were carefully diluted by ice-cold water (100 ml). Theprecipitated 5-aroyl-4-amino-2-arylaminothiazoles 5 were collected byfiltration and purified by crystallization or column chromatography onsilica gel. A few typical results in the preparation of thiazole 5 isgiven below.

No Ar Ar′ Yield % 5a C₆H₅ C₆H₅ 65-68 5b 4-Cl—C₆H₄ C₆H₅ 73-78 5c4-Me-C₆H₅ C₆H₅ 68-72 5d 4-OMe C₆H₅ C₆H₅ 67-71

3. Cell Viability Assay

MTT assay was used to determine the number of viable cells upon drugaddition. Cells were seeded in microtitre plates (generally 5×10³ cellsper well) and were incubated with different concentrations of thecytotoxic agents for 48 h. Subsequently, 100 μl of MTT solution (0.6mg/ml) was added per well and incubated at 37° C. for additional 2 h.The amount of formazan salt was quantified in quadruplicates byrecording the absorbance at 570 nm using a Biorad Plate reader. Thegrowth inhibition constants (IC₅₀) were calculated from thesemi-logarithmic dose response plots using the nonlinear regressionprogram Origin. All the experiments were done for at least three times.

4. Microtubule and Tubulin Preparation

Microtubular protein was prepared from goat brains by two cycles oftemperature dependent assembly-disassembly process in PEM buffer (100 mMPIPES, pH 6.9, 1 mM MgCl₂ & 1 mM EGTA) with 1 mM GTP at 37° C. For thepolymerization experiments it was followed by one more cycle in PEM.Tubulin was purified from 2×MTP using glutamate buffer for assembly.

5. Polymerization Assay

MTP polymerization, in the presence or absence of DAT1, was measured bythe time course of the turbidity at 37° C. at 345 nm. A Shimadzu UV-1601double beam spectrophotometer fitted with a temperature-controlledcirculating water bath was used for this purpose.

6. Immunofluorescence Assay

HeLa cells were incubated with the drug for 24 h at 37° C., washed withPBS and fixed with 4% paraformaldehyde at 4° C. Subsequently, they werewashed and permeabilized with 0.2% Triton X-100 in PBS or 20 min at 37°C. Microtubules were stained by a mouse monoclonal antibody againstβ-tubulin in 1:100 dilution followed by a Rhodamine conjugated goatantimouse antibody in 1:50 dilution and were observed by a Nikon EclipseTE300 microscope.

7. Tubulin Binding

Emission spectra of DAT1 in presence of tubulin were recorded from 400nm to 600 nm using an excitation wavelength of 374 nm. Excitation andemission bandpasses are 2.5 nm each and the fluorescence values recordedare uncorrected. All fluorescence measurements were performed in aPerkin-Elmer model LS50B Luminescence spectrometer.

The binding parameters of DAT1 binding to tubulin were measured fromfluorescence data by the standard Scatchard analysis. The bindingconstants and stoichiometries were determined from Scatchard plot using2 μM tubulin and varying DAT1 over 0.2-20 μM. Fluorescence values wererecorded at 450 nm using an excitation wavelength of 350 nm to reducethe absorbance of DAT1. Inner filter effect corrections were performedto minimize the effect of high absorbance of the fluorophore.

TABLE 1 Cytotoxic activity of DAT1 against different tumour cell linesCell Lines IC₅₀ values (Source) DAT1 Paclitaxel Vinblastine ColchicineHCT 116 (Human 0.3 μM 0.012 μM 0.005 μM colon) HeLa (Human 0.054 μM0.034 μM 0.001 μM cervix) L-929 (Mouse 1 μM 0.3 μM connective tissue)CaSki (Human 0.2 μM 0.02 μM 0.25 μM cervix) SW 620 (Human 0.2 μM 0.007μM 0.2 μM colon) SiHa (Human 5 μM 1.1 μM  2.5 μM cervix) MES-SA/Dx5 0.35μM 8.6 μM 0.73 μM (Human uterus)

Different concentrations of DAT1, paclitaxel and vinblastine orcolchicine were incubated at 37° C. with the different cell lines. After48 hours, drug containing media were removed and MTT assay were done asdescribed in the examples IC₅₀ values (growth inhibition constants) werecalculated using the nonlinear regression program Origin. The average ofthree experiments is shown.

TABLE 2 Physical data of DAT1 Ar^(′) C₆H₅— Ar 4-CH₃O—C₆H₄— m.p ° C.205-6 Yield % 67-71% IR (KBr) 3344, 3179, 1600, 1557, 1513, 1459, 1366,1251, 1169, 1108, 1058 cm⁻¹ 1025, 912, 743, 705 ¹H NMR δ 3.82 (s, 3H),6.9-7.72 (m, 9H), 8.15 (br, 2H) MS m/z EIMS: 325 (24, M⁺), 323 (11), 248(3), 220 (5), 165 (7) (%) 149 (15), 148 (8), 134 (11), 133 (16), 122(16), 105 (58), 78 (26), 77 (100)

1. A method of microtubule inhibition comprising: administering adiaminoketothiazole (DAT1) having a formula:

wherein Ar is 4-OMe-C₆H₅ and Ar′ is C₆H₅; and wherein the DAT1 compoundis a cytotoxic agent.
 2. The method of microtubule inhibition of claim1, wherein the DAT1 distorts a microtubule in a cancer cell and inhibitsin vitro assembly of a microtubular protein.
 3. The method ofmicrotubule inhibition of claim 1, wherein the DAT1 arrests mitosisthereby treating a disease where unwanted cell division is involved. 4.The method of microtubule inhibition of claim 1, wherein the DAT1inhibits microtubule polymerization thereby shifting tubulin-microtubuleequilibrium.
 5. The method of microtubule inhibition of claim 1, whereinthe DAT1 binds to tubulin with high affinity.
 6. The method ofmicrotubule inhibition of claim 5, further comprising using fluorescenceto bind the DAT1 to the tublin.
 7. The method of microtubule inhibitionof claim 2, wherein the cancer cell is a HeLa cancer cell.